Distinctive ERK and p38 signaling in remote and infarcted myocardium during post-MI remodeling in the mouse

Abstract

Global activation of MAP kinases has been reported in both human and experimental heart failure. Chronic remodeling of the surviving ventricular wall after myocardial infarction (MI) involves both myocyte loss and fibrosis; we hypothesized that this cardiomyopathy involves differential shifts in pro- and anti-apoptotic MAP kinase signaling in cardiac myocyte (CM) and non-myocyte. Cardiomyopathy after coronary artery ligation in mice was characterized by echocardiography, ex vivo Langendorff preparation, histologic analysis and measurements of apoptosis. Phosphorylation (activation) of signaling molecules was analyzed by Western blot, ELISA and immunohistochemistry. Post-MI remodeling involved dramatic changes in the phosphorylation of both stress-activated MAP (SAP) kinase p38 as well as ERK, a known mediator of cell survival, but not of SAP kinase JNK or the anti-apoptotic mediator of PI3K, Akt. Phosphorylation of p38 rose early after MI in the infarct, whereas a more gradual rise in the remote myocardium accompanied a rise in apoptosis in that region. In both areas, ERK phosphorylation was lowest early after MI and rose steadily thereafter, though infarct phosphorylation was consistently higher. Immunostaining of p-ERK localized to fibrotic areas populated primarily by non-myocytes, whereas staining of p38 phosphorylation was stronger in areas of progressive CM apoptosis. Relative segregation of CMs and non-myocytes in different regions of the post-MI myocardium revealed signaling patterns that imply cell type-specific changes in pro- and anti-apoptotic MAP kinase signaling. Prevention of myocyte loss and of LV remodeling after MI may therefore require cell type-specific manipulation of p38 and ERK activation.

Fig. 1

A: Representative Western blots indicating phosphorylation (i.e., activation) of p38 MAPK in the remote myocardium during post-MI remodeling. Changes in p38α (white bars) and p38γ (gray bars) were measured via ELISA of tissue lysates from remote (B) or infarct/borderzone myocardium (C). n = 4–9, *P < 0.05, week 8 and 12 versus week 1.

Fig. 2

Representative Western blots (A) and ELISA (B,C) indicated increases in ERK phosphorylation (i.e., activation) during chronic post-MI remodeling that were consistently greater in the infarcted/borderzone region than in the remote myocardium. n = 4–9, *P < 0.05 versus normal control.

Fig. 3

Myocardial JNK (white bars) and Akt (gray bars) activation in the remote (A) and in the infarct/borderzone (B) myocardium after myocardial infarction. n = 4–9, *P < 0.05 vs. normal control.

Fig. 4

Immunofluorescent co-staining of phospho-p38 (red, A,B,D,E) and phospho-ERK (red, C,F) together with myosin heavy chain staining using MF-20 (green). Nuclear staining for phospho-p38 was observed in both myocytes (arrows) and non-myocytes (arrowheads), whereas phospho-ERK staining, even in fibrotic areas of the remote myocardium, predominated in non-myocytes (arrowheads). Nuclei are counterstained with DAPI; cardiac non-myocytes appear black. A–C: 400 ×; D–F: 1,600 ×.

Fig. 5

A: Representative Western blot of lystaes from cardiac myocytes and fibroblasts isolated from the same mouse hearts and cultured for 48 h in vitro. Note the identification of p38 activation but not ERK activation in isolated myocytes, both with and without adrenergic stimulation with norepinepherine. In contrast, both ERK and p38 MAPK were activated in cardiac fibroblasts, particularly with adrenergic stimulation. B: Simultaneous adrenergic stimulation with epinephrine (EPI) and blockade of beta receptors with propranolol (PROP) unmasked an apparent alpha adrenergic-induced phosphorylation of ERK in isolated myocytes compared to myocytes treated with vehicle and propranolol alone (VC PROP).